Luca Mazzoni

UDP-glucose enhances outward K+ currents necessary for cell differentiation and stimulates cell migration by activating the GPR17 receptor in oligodendrocyte precursors

In the developing and mature central nervous system, NG2 expressing cells comprise a population of cycling oligodendrocyte progenitor cells (OPCs) that differentiate into mature, myelinating oligodendrocytes (OLGs). OPCs are also characterized by high motility and respond to injury by migrating into the lesioned area to support remyelination. K+ currents in OPCs are developmentally regulated during differentiation. However, the mechanisms regulating these currents at different stages of oligodendrocyte lineage are poorly understood. Here we show that, in cultured primary OPCs, the purinergic G‐protein coupled receptor GPR17, that has recently emerged as a key player in oligodendrogliogenesis, crucially regulates K+ currents. Specifically, receptor stimulation by its agonist UDP‐glucose enhances delayed rectifier K+ currents without affecting transient K+ conductances. This effect was observed in a subpopulation of OPCs and immature pre‐OLGs whereas it was absent in mature OLGs, in line with GPR17 expression, that peaks at intermediate phases of oligodendrocyte differentiation and is thereafter downregulated to allow terminal maturation. The effect of UDP‐glucose on K+ currents is concentration‐dependent, blocked by the GPR17 antagonists MRS2179 and cangrelor, and sensitive to the K+ channel blocker tetraethyl‐ammonium, which also inhibits oligodendrocyte maturation. We propose that stimulation of K+ currents is responsible for GPR17‐induced oligodendrocyte differentiation. Moreover, we demonstrate, for the first time, that GPR17 activation stimulates OPC migration, suggesting an important role for this receptor after brain injury. Our data indicate that modulation of GPR17 may represent a strategy to potentiate the post‐traumatic response of OPCs under demyelinating conditions, such as multiple sclerosis, stroke, and brain trauma.

Novel insights on the relationship between T-tubular defects and contractile dysfunction in a mouse model of hypertrophic cardiomyopathy

Abnormalities of cardiomyocyte Ca2 + homeostasis and excitation–contraction (E–C) coupling are early events in the pathogenesis of hypertrophic cardiomyopathy (HCM) and concomitant determinants of the diastolic dysfunction and arrhythmias typical of the disease. T-tubule remodelling has been reported to occur in HCM but little is known about its role in the E–C coupling alterations of HCM. Here, the role of T-tubule remodelling in the electro-mechanical dysfunction associated to HCM is investigated in the Δ160E cTnT mouse model that expresses a clinically-relevant HCM mutation. Contractile function of intact ventricular trabeculae is assessed in Δ160E mice and wild-type siblings. As compared with wild-type, Δ160E trabeculae show prolonged kinetics of force development and relaxation, blunted force-frequency response with reduced active tension at high stimulation frequency, and increased occurrence of spontaneous contractions. Consistently, prolonged Ca2 + transient in terms of rise and duration are also observed in Δ160E trabeculae and isolated cardiomyocytes. Confocal imaging in cells isolated from Δ160E mice reveals significant, though modest, remodelling of T-tubular architecture. A two-photon random access microscope is employed to dissect the spatio-temporal relationship between T-tubular electrical activity and local Ca2 + release in isolated cardiomyocytes. In Δ160E cardiomyocytes, a significant number of T-tubules (> 20%) fails to propagate action potentials, with consequent delay of local Ca2 + release. At variance with wild-type, we also observe significantly increased variability of local Ca2 + transient rise as well as higher Ca2 +-spark frequency. Although T-tubule structural remodelling in Δ160E myocytes is modest, T-tubule functional defects determine non-homogeneous Ca2 + release and delayed myofilament activation that significantly contribute to mechanical dysfunction.

Ranolazine Prevents Phenotype Development in a Mouse Model of Hypertrophic Cardiomyopathy.

Background— Current therapies are ineffective in preventing the development of cardiac phenotype in young carriers of mutations associated with hypertrophic cardiomyopathy (HCM). Ranolazine, a late Na+ current blocker, reduced the electromechanical dysfunction of human HCM myocardium in vitro. Methods and Results— To test whether long-term treatment prevents cardiomyopathy in vivo, transgenic mice harboring the R92Q troponin-T mutation and wild-type littermates received an oral lifelong treatment with ranolazine and were compared with age-matched vehicle-treated animals. In 12-months-old male R92Q mice, ranolazine at therapeutic plasma concentrations prevented the development of HCM-related cardiac phenotype, including thickening of the interventricular septum, left ventricular volume reduction, left ventricular hypercontractility, diastolic dysfunction, left-atrial enlargement and left ventricular fibrosis, as evaluated in vivo using echocardiography and magnetic resonance. Left ventricular cardiomyocytes from vehicle-treated R92Q mice showed marked excitation–contraction coupling abnormalities, including increased diastolic [Ca2+] and Ca2+ waves, whereas cells from treated mutants were undistinguishable from those from wild-type mice. Intact trabeculae from vehicle-treated mutants displayed inotropic insufficiency, increased diastolic tension, and premature contractions; ranolazine treatment counteracted the development of myocardial mechanical abnormalities. In mutant myocytes, ranolazine inhibited the enhanced late Na+ current and reduced intracellular [Na+] and diastolic [Ca2+], ultimately preventing the pathological increase of calmodulin kinase activity in treated mice. Conclusions— Owing to the sustained reduction of intracellular Ca2+ and calmodulin kinase activity, ranolazine prevented the development of morphological and functional cardiac phenotype in mice carrying a clinically relevant HCM-related mutation. Pharmacological inhibitors of late Na+ current are promising candidates for an early preventive therapy in young phenotype-negative subjects carrying high-risk HCM-related mutations.

Isolation and functional characterization of human ventricular cardiomyocytes from fresh surgical samples.

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models. Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method. The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

R4496C RyR2 mutation impairs atrial and ventricular contractility

A ryanodine receptor 2 mutation associated with catecholaminergic polymorphic ventricular tachycardia renders cardiomyocytes incapable of mediating a positive inotropic response.

Regulation of intracellular Na+ in health and disease: pathophysiological mechanisms and implications for treatment

Transmembrane sodium (Na+) fluxes and intracellular sodium homeostasis are central players in the physiology of the cardiac myocyte, since they are crucial for both cell excitability and for the regulation of the intracellular calcium concentration. Furthermore, Na+ fluxes across the membrane of mitochondria affect the concentration of protons and calcium in the matrix, regulating mitochondrial function. In this review we first analyze the main molecular determinants of sodium fluxes across the sarcolemma and the mitochondrial membrane and describe their role in the physiology of the healthy myocyte. In particular we focus on the interplay between intracellular Ca2+ and Na+. A large part of the review is dedicated to discuss the changes of Na+ fluxes and intracellular Na+ concentration([Na+]i) occurring in cardiac disease; we specifically focus on heart failure and hypertrophic cardiomyopathy, where increased intracellular [Na+]i is an established determinant of myocardial dysfunction. We review experimental evidence attributing the increase of [Na+]i to either decreased Na+ efflux (e.g. via the Na+/K+ pump) or increased Na+ influx into the myocyte (e.g. via Na+ channels). In particular, we focus on the role of the “late sodium current” (INaL), a sustained component of the fast Na+ current of cardiac myocytes, which is abnormally enhanced in cardiac diseases and contributes to both electrical and contractile dysfunction. We analyze the pathophysiological role of INaL enhancement in heart failure and hypertrophic cardiomyopathy and the consequences of its pharmacological modulation, highlighting the clinical implications. The central role of Na+ fluxes and intracellular Na+ physiology and pathophysiology of cardiac myocytes has been highlighted by a large number of recent works. The possibility of modulating Na+ inward fluxes and [Na+]i with specific INaL inhibitors, such as ranolazine, has made Na+a novel suitable target for cardiac therapy, potentially capable of addressing arrhythmogenesis and diastolic dysfunction in severe conditions such as heart failure and hypertrophic cardiomyopathy.

Pathogenesis of Hypertrophic Cardiomyopathy is Mutation Rather Than Disease Specific: A Comparison of the Cardiac Troponin T E163R and R92Q Mouse Models

Background In cardiomyocytes from patients with hypertrophic cardiomyopathy, mechanical dysfunction and arrhythmogenicity are caused by mutation‐driven changes in myofilament function combined with excitation‐contraction (E‐C) coupling abnormalities related to adverse remodeling. Whether myofilament or E‐C coupling alterations are more relevant in disease development is unknown. Here, we aim to investigate whether the relative roles of myofilament dysfunction and E‐C coupling remodeling in determining the hypertrophic cardiomyopathy phenotype are mutation specific. Methods and Results Two hypertrophic cardiomyopathy mouse models carrying the R92Q and the E163R TNNT2 mutations were investigated. Echocardiography showed left ventricular hypertrophy, enhanced contractility, and diastolic dysfunction in both models; however, these phenotypes were more pronounced in the R92Q mice. Both E163R and R92Q trabeculae showed prolonged twitch relaxation and increased occurrence of premature beats. In E163R ventricular myofibrils or skinned trabeculae, relaxation following Ca2+ removal was prolonged; resting tension and resting ATPase were higher; and isometric ATPase at maximal Ca2+ activation, the energy cost of tension generation, and myofilament Ca2+ sensitivity were increased compared with that in wild‐type mice. No sarcomeric changes were observed in R92Q versus wild‐type mice, except for a large increase in myofilament Ca2+ sensitivity. In R92Q myocardium, we found a blunted response to inotropic interventions, slower decay of Ca2+ transients, reduced SERCA function, and increased Ca2+/calmodulin kinase II activity. Contrarily, secondary alterations of E‐C coupling and signaling were minimal in E163R myocardium. Conclusions In E163R models, mutation‐driven myofilament abnormalities directly cause myocardial dysfunction. In R92Q, diastolic dysfunction and arrhythmogenicity are mediated by profound cardiomyocyte signaling and E‐C coupling changes. Similar hypertrophic cardiomyopathy phenotypes can be generated through different pathways, implying different strategies for a precision medicine approach to treatment.

Late sodium current inhibitors to treat exercise‐induced obstruction in hypertrophic cardiomyopathy: an in vitro study in human myocardium

In 30–40% of hypertrophic cardiomyopathy (HCM) patients, symptomatic left ventricular (LV) outflow gradients develop only during exercise due to catecholamine‐induced LV hypercontractility (inducible obstruction). Negative inotropic pharmacological options are limited to β‐blockers or disopyramide, with low efficacy and tolerability. We assessed the potential of late sodium current (INaL)‐inhibitors to treat inducible obstruction in HCM.